Measurement of plasma concentrations of three drugs

The plasma concentration of each drug was measured by LC-MS/MS based on the ESI (electrospray ionization) method. The LC-MS/MS system consisted of a high-performance liquid chromatograph LC-30AD system (Shimadzu, Kyoto, Japan) and an AB Sciex QTRAP 6500 tandem mass spectrometer (Framingham, MA, USA). Capcell Pak C18 MGII (particle size 5 μm, inner diameter 2.0 mm x 50 mm; Osaka Soda, Osaka, Japan) was used as the separation column. To measure the concentration of SVA, solvent A (water containing 0.063% ammonium formate) and solvent B (acetonitrile) were used as the mobile phase. The gradient conditions were maintained at 20% solvent B for 1.5 min, increased from 20% to 95% solvent B in 1.50 min, held at 95% for 1.0 min, decreased back to 20% within 0.01 min, and held at 20% for 1.49 min. To measure the concentration of LEV and CBZ, solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile) were used as the mobile phase. The gradient conditions of LEV were increased from 5% to 60% solvent B in 1.50 min, sharply increased to 95% within 0.01 min, held at 95% for 1.49 min, decreased back to 5% within 0.01 min, and held at 5% for 1.49 min. The gradient conditions of CBZ were increased from 30% to 95% solvent B in 2.00 min, held at 95% for 1.00 min, decreased back to 30% within 0.01 min, and held at 30% for 1.49 min.